Kidney perfusion solution containing nitric oxide donor

ABSTRACT

A kidney perfusion solution which includes at least one gluconate salt; glutathione; a nitric oxide donor chemical and a chemical inhibitor selective for the inducible isoform of the enzyme nitric oxide synthase; optionally further containing a reagent that causes the reduction of oxidized glutathione. A process for preserving a kidney for transplantation is also disclosed, which includes perfusing the kidney with an amount of a nitric oxide donor chemical in an amount sufficient to mimic enzymatic production of NO by NOS3 or NOS1, while preventing generation of an excessive amount of nitric oxide by the NOS2 isoform of nitric oxide synthase, with or without a reagent that causes the reduction of oxidized glutathione. The invention also includes a process wherein a deceased donor&#39;s body is perfused with a solution containing an NO donor, with or without a reagent that causes the reduction of oxidized glutathione, prior to removal of the organs for transplantation.

BACKGROUND OF THE INVENTION

[0001] This invention relates to kidney perfusion solutions and a methodfor increasing the viability of perfused kidneys prior totransplantation.

[0002] Kidneys must be preserved for a period of at least 5 hours priorto transplantation to obtain proper pathology assessment of thesuitability of the organ for transplantation. Lack of properpreservation leads to degradation of organ function due to thrombosis(blood clotting), ischemia (lack of oxygen), or ischemia followed byreperfusion (the restoration of blood flow upon transplantation). Theseevents bring about inflammation, cell death, and eventually failure ofthe organ. The preferred method for preserving kidneys is pulsatilepreservation.

[0003] Pulsatile kidney preservation is a process in which the renalartery is connected to a kidney perfusion machine in order to simulatethe normal process by which nutrients are supplied to the kidney. Asolution is continuously perfused through a closed circuit whichincludes the kidney, which is typically maintained at 5 degrees C. Inorder for pulsatile preservation to be an effective method forpreservation of “extended criteria” organs (i.e., organs which are lessoptimal than those currently accepted for transplantion), the technicianneeds to monitor closely not just perfusion pressure, flow, and vascularresistance, but also the organ's chemistries, including base excess,oxygen saturation, calcium, potassium, hematocrit, pO₂, pH, andbicarbonate. This method has become the standard of care for kidneytransplantation, due to its efficacy and cost effectiveness. See Lightet al., “Immediate function and cost comparison between static andpulsatile preservation in kidney recipients,” Clin. Transplantation233-236 (1996).

[0004] Although pulsatile perfusion of kidneys is superior to staticpreservation methods, pulsatile perfusion suffers from severaldrawbacks. It requires continuous monitoring and correction ofchemistries as well as pressure and flow in order to be optimal, andthus the process is time- and labor-intensive (and hence expensive).Moreover, organ perfusion requires extensive expertise, and results canvary from perfusionist to perfusionist. Another problem that is observedwith current kidney perfusion solutions is the rapid oxidation ofglutathione, a key component of current kidney perfusion solutions thatserves as an antioxidant. Finally, pulsatile preservation has onlyproven marginally effective at preserving organs from “extendedcriteria” donors.

[0005] Nitric oxide (NO) can have both beneficial and detrimentaleffects in the kidney. A low level of NO, produced by the so-calledconstitutive nitric oxide synthases (NOS), found in endothelial cells(NOS3; ecNOS) or neurons and some other cell types (NOS1; nNOS), appearsto be necessary for the maintenance of homeostasis in the kidney. See,for example, Kone et al., “Biosynthesis and homeostatic roles of nitricoxide in the normal kidney,” Am. J. Physiol. F561-578 (1997) andRadermacher et al., “Importance of NO/EDRF for glomerular and tubularfunction: studies in the isolated perfused rat kidney,” Kidney Int.1549-1559 (1992). However, it has also been reported that high levels ofNO produced by the inducible nitric oxide synthase (NOS2) during theprocess of kidney damage and/or transplantation are detrimental to thekidney, Klahr et al., “Renal disease: The two faces of nitric oxide,”Lab. Invest. 1-3 (1995). All three isoforms of NOS (NOS1, NOS2, andNOS3) are found in the kidney, though not all of their functions areknown. See, for example, Kone et al, “Localization and regulation ofnitric oxide synthase isoforms in the kidney,” Semin. Nephrol. 230-241(1999).

[0006] Transplant patients are often treated with immunosuppressiveagents such as cyclosporine A in order to prevent transplant rejection.Such immunosuppressive agents can suppress nitric oxide production inthe normal kidney, and thus can suppress certain kidney functions. SeeBloom et al, “An experimental study of altered nitric oxide metabolismas a mechanism of cyclosporin-induced renal vasoconstriction,” Br. J.Surg. 195-198 (1995) and Gaston et al., “Cyclosporine inhibits the renalresponse to L-arginine in human kidney transplant recipients,” J. Am.Soc. Nephrol. 1426-1433 (1995).

[0007] Nitric oxide donor chemicals are known, and have proven safe andefficacious at modulating various physiological and pathologicalparameters associated with the presence of nitric oxide. The nitricoxide donor chemical S-nitrosoglutathione (GSNO) has been reported toreduce platelet aggregation both in vitro and in vivo, to reduce acutemyocardial infarction and unstable angina, to be of therapeutic benefitto patients with the HELLP syndrome and in fetal pre-eclampsia, toinhibit platelet activity in patients undergoing percutaneoustransluminal coronary angioplasty (PTCA) or saphenous vein grafts, andto inhibit vasospasm in human coronary arteries. GSNO was found tosuppress thrombosis in a porcine model of balloon angioplasty andsubsequent endovascular radiation, see Vodovotz et al.,“S-nitrosoglutathione reduces non-occlusive thrombosis rate followingballoon overstretch injury and intracoronary radiation of porcinecoronary arteries,” 48 Int. J. Radiat. Oncol. Biol. Phys. 1167-1174(2000).

[0008] Sodium nitroprusside, another nitric oxide donor chemical, hasrecently been suggested for addition to static liver perfusion solution.Rodriguez et al., “Role of sodium nitroprusside in the improvement ofrat liver preservation in University of Wisconsin solution: A study inthe isolated perfused liver model,” 87 J.Surg.Res. 201-208 (1999).

[0009] An object of the present invention is to preserve kidneys thathave been taken from donors prior to transplantation into a recipient,by providing the low levels of nitric oxide necessary for optimal kidneyfunction (essentially substituting for reduced levels or activity ofNOS1 or NOS3), while inhibiting over-exuberant enzymatic production ofNO from NOS2 that can damage the kidney.

[0010] A feature of the present invention is the presence of a nitricoxide donor chemical and a chemical inhibitor selective for theinducible isoform of nitric oxide synthase (NOS2) in a kidney pulsatileperfusion solution.

[0011] An advantage of the present invention is that it should permitthe transplantation of kidneys from donors whose kidneys are lessoptimal than those accepted currently (“extended criteria” organs).

[0012] Use of a particular nitric oxide donor chemical, GSNO, would havean additional advantage in that it would generate oxidized glutathionein addition to nitric oxide. This oxidized glutathione, if givensimultaneously with an additional reagent that could reduce glutathioneback to its bioactive form (such as the enzyme glutathione reductase aswell as the necessary cofactors for the activity of this enzyme) wouldyield higher levels of bioactive glutathione.

SUMMARY OF THE INVENTION

[0013] In one aspect, the present invention is directed to a kidneypulsatile perfusion solution comprising at least one gluconate salt,glutathione, a nitric oxide donor chemical, and a chemical inhibitorselective for the inducible isoform of nitric oxide synthase.

[0014] In another aspect, the present invention relates to an improvedprocess for perfusing a kidney with an amount of a nitric oxide donorchemical in an amount sufficient to mimic enzymatic production of nitricoxide by the beneficial enzymes NOS1 or NOS3, while preventinggeneration of an excessive amount of nitric oxide by NOS2.

[0015] In a third aspect, the present invention is directed to a methodfor increasing the availability of bioactive glutathione in a perfusionsolution by including an additional reagent to reduce glutathione backto its bioactive form.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a is a plot of vascular resistance vs. time for porcinekidneys perfused with either a control perfusion solution, the controlperfusion solution additionally containing a nitric oxide donor chemicalS-nitrosoglutathione (GSNO), or the control perfusion solutionadditionally containing GSNO and N-omega-imino ethyl lysine (L-NIL), achemical inhibitor selective for the inducible isoform of the enzymenitric oxide synthase.

[0017]FIG. 2 is a is a plot of flow vs. time for porcine kidneysperfused with either a control perfusion solution, the control perfusionsolution additionally containing GSNO, or the control perfusion solutionadditionally containing GSNO and L-NIL.

[0018]FIG. 3 is a is a plot of base excess vs. time for porcine kidneysperfused with either a control perfusion solution, the control perfusionsolution additionally containing GSNO, or the control perfusion solutionadditionally containing GSNO and L-NIL.

[0019]FIG. 4 is a is a plot of oxygen saturation vs. time for porcinekidneys perfused with either a control perfusion solution, the controlperfusion solution additionally containing GSNO, or the controlperfusion solution additionally containing GSNO and L-NIL.

[0020]FIG. 5 is a is a plot of calcium vs. time for porcine kidneysperfused with either a control perfusion solution, the control perfusionsolution additionally containing GSNO, or the control perfusion solutionadditionally containing GSNO and L-NIL.

[0021]FIG. 6 is a plot of hematocrit vs. time for porcine kidneysperfused with either a control perfusion solution, the control perfusionsolution additionally containing GSNO, or the control perfusion solutionadditionally containing GSNO and L-NIL.

[0022]FIG. 7 is a is a plot of pO₂ @ 8° C. vs. time for porcine kidneysperfused with either a control perfusion solution, the control perfusionsolution additionally containing GSNO, or the control perfusion solutionadditionally containing GSNO and L-NIL.

[0023]FIG. 8 is a plot of pH @ 8° C. vs. time for porcine kidneysperfused with either a control perfusion solution, the control perfusionsolution additionally containing GSNO, or the control perfusion solutionadditionally containing GSNO and L-NIL.

[0024]FIG. 9 is a plot of potassium vs. time for porcine kidneysperfused with either a control perfusion solution, the control perfusionsolution additionally containing GSNO, or the control perfusion solutionadditionally containing GSNO and L-NIL.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] A “nitric oxide donor chemical” is a chemical that can decomposeinto nitric oxide and a residue of the nitric oxide donor chemical,which may promptly react with one or more compounds found in the mediumin which the nitric oxide chemical donor decomposes. Nitric oxide donorchemicals include, but are not limited to, S-nitrosoglutathione(molecular weight 336.3), S-nitrosoalbumin (molecular weightapproximately 66,000), S-nitroso-N-acetyl-D,L-penicillamine (molecularweight 220.3), diethylamine-NONOate(1,1-Diethyl-2-hydroxy-2-nitroso-hydrazine sodium; molecular weight155.13), diethylenetriamine-NONOate(2,2′-(Hydroxynitrosohydrazono)bis-ethanimine; molecular weight 163.18),and spermine-NONOate(N-(2-Aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine;molecular weight 262.36), and their pharmaceutically acceptable salts,esters and derivatives. Such nitric oxide chemical donors can be storedas powders for extended periods, and decompose spontaneously when placedin solution to yield nitric oxide and a nitric oxide donor chemicalresidue. See Keefer, L. K. et al, “‘NONOates’ (1-substituteddiazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitricoxide dosage forms,” Methods Enzymol. 281-293 (1996); and Stamler, J.S., “S-nitrosothiols and the bioregulatory actions of nitrogen oxidesthrough reactions with thiol groups,” Curr. Top. Microbiol. Immunol.19-36 (1995), the disclosures of which are each incorporated byreference herein in their entirety.

[0026] S-nitrosoglutathione and S-nitrosoalbumin are particularlypreferred as nitric oxide donor chemicals because they decompose, eitherdirectly or indirectly, to yield NO and a residue compound, oxidizedglutathione and albumin, respectively, both of which are components ofconventional kidney pulsatile preservation solutions.

[0027] Any conventional kidney pulsatile perfusion solution may beemployed in the present invention. Kidney pulsatile perfusion solutionstypically contain the following reagents, in various proportions: sodiumgluconate, hydroxyethyl starch, human serum albumin, KH₂PO₄, glucose,glutathione, adenosine, magnesium gluconate, adenine, ribose, calciumchloride, Hepes (N-[2-Hydroxyethyl]piperazine-N0-[2-ethanesulfonicacid]), mannitol, penicillin G, dexamethasone, and insulin. Table 1below lists the components of an illustrative kidney perfusion solution:TABLE 1 Kidney Perfusion Solution Formulation Concentration PreferredComponent Range Concentration Sodium 60-80  millimolar 65-75  millimolarGluconate Potassium 5-15  millimolar 8-12  millimolar GluconateMagnesium 2-9   millimolar 4-6   millimolar Gluconate Hydroxyethyl40-60  g/liter 45-55  g/liter Starch KH₂PO₄ 10-20  millimolar 13-17 millimolar Glutathione 2-7   millimolar 2-4   millimolar Hepes 5-15 millimolar 8-12  millimolar Adenine 2-10  millimolar 3-7   millimolarRibose 2-10  millimolar 3-7   millimolar CaCl₂ 0.2-0.8 millimolar0.4-0.6 millimolar Allopurinol 0.5-2   millimolar 0.5-1.5 millimolarInsulin 30-50  Units/liter 35-45  Units/liter Dexamethasone 6-10  mg/ml7-9   mg/ml

[0028] The kidney perfusion solution of the present invention includesat least one nitric oxide donor chemical in an amount which is effectiveto preserve a kidney prior to transplantation. A preferred nitric oxidedonor chemical concentration range is 10 to 500 micromolar, still morepreferably 20 to 125 micromolar.

[0029] Without intending to be bound by theory, the inventors currentlybelieve that the addition of a nitric oxide donor chemical to anotherwise conventional kidney perfusion solution will increase thebioavailability of NO, and thus enhance the viability and increasecertain functions of kidneys maintained on perfusion machines inpreparation for transplantation. Kidneys thus treated will have agreater chance of maintaining function in the patient aftertransplantation. The inclusion of a nitric oxide donor chemical in organpulsatile perfusion solutions should therefore permit thetransplantation of kidneys from donors who are more marginal than thoseaccepted currently.

[0030] Most nitric oxide donor chemicals decompose spontaneously in thetype of solutions used to perfuse kidneys, and nitric oxide donorchemicals with very different rates of decomposition are available.Proper selection of the nitric oxide donor chemical will permitappropriate control over the rate of NO produced over time (“flux”),depending on whether short-term or prolonged bioavailability of NO isdesired.

[0031] In a preferred embodiment, the nitric oxide donor chemical yieldsboth NO and an antioxidant upon decomposition, either directly orindirectly. These antioxidants will also increase the viability andfunction of kidneys maintained on perfusion machines in preparation fortransplantation. More particularly, the antioxidant will scavengeoxidized free radicals, which are known to cause organ damage. Thus, aparticularly preferred nitric oxide chemical donor isS-nitrosoglutathione (GSNO), which will decompose to yield NO andoxidized glutathione; oxidized glutathione will then be reduced by anenzyme either present in the kidney [glutathione reductase; see Di Ilioet al., “Glutathione peroxidase and glutathione reductase activities incancerous and non-cancerous human kidney tissues,” Cancer Lett. 19-23(1995)] or by the addition of glutathione peroxidase to the perfusionsolution. Glutathione has been reported to have beneficial effects inisolated perfused kidneys or in ischemic kidneys. See McCoy et al.,“Oxidant stress following renal ischemia: Changes in the glutathioneredox ratio,” Kidney Int. 812-817 (1988) and Boudjema et al., “Changesin glutathione concentration in hypothermically perfused dog kidneys,”J. Lab. Clin. Med. 131-137 (1991). As described above, glutathione isused as a conventional component of current formulations of kidneypulsatile perfusion solutions. Addition of a reagent that could reduceglutathione back to its bioactive form (e.g. glutathione reductase andits requisite cofactors) would serve to increase bioactive glutathioneboth as a byproduct of decomposition of S-nitrosoglutathione but alsofrom the glutathione already present in the pulsatile perfusionsolution.

[0032] The kidney pulsatile solution may also contain a potentantioxidant to scavenge superoxide radical or an inhibitor whichsuppresses superoxide production, since superoxide can react with nitricoxide and thereby reduce the bioavailability of nitric oxide. Copper,zinc superoxide dismutase and manganese superoxide dismutase areillustrative superoxide scavengers, while ethinylestradiol may be citedas a superoxide inhibitor.

[0033] The kidney pulsatile solution of the present invention alsocontains a chemical inhibitor selective for the inducible isoform of theenzyme nitric oxide synthase (NOS2; iNOS). Illustrative nitric oxidesynthase inhibitors include but are not limited to N-omega-imino ethyllysine (L-NIL; molecular weight 187.2), N-omega-imino ethyl-L-ornithine(L-NIO,; molecular weight 173.2), ([aminomethyl] benzyl)acetamidine(1400 W; molecular weight 177.3), S-(2-aminoethyl)-isothiourea (AET;molecular weight 119.2), aminoguanidine hydrochloride (molecular weight74.1), S-ethyl-isothiourea (SEITU; molecular weight 104.2), andS-methyl-isothiourea (SMT; molecular weight 180.3), and theirpharmaceutically acceptable salts, esters and derivatives.

[0034] The nitric oxide synthase inhibitor should be present in anamount that is effective to ensure that a harmful excess of NO is notgenerated during perfusion. A preferred concentration range is 10 to5,000 micromolar. A still more preferred range is 500 to 2000micromolar.

[0035] Again without intending to be bound by theory, the inventorscurrently believe that addition of a selective nitric oxide synthaseinhibitor will reduce transplant rejection because induction of NOS2 intransplanted organs such as the liver has been shown to correlate withtransplant rejection. See Ioannidis et al., “Evidence for increasednitric oxide production after liver transplantation in humans,”Transplantation 1293-1297 (1995). Furthermore, the NOS2 inhibitor shouldimprove kidney function by reducing damage to the kidneys mediated byover-exuberant production of NO (see, for example, Klahr et al., “Renaldisease: The two faces of nitric oxide,” Lab. Invest. 1-3 [1995]).

[0036] The nitric oxide donor chemical and the chemical NOS2 inhibitor,with or without an additional reagent that could reduce glutathione backto its bioactive form and thereby increase bioactive glutathione, may beadded to a conventional kidney perfusion solution as a powder or liquidusing conventional mixing techniques and apparatus. These additives maybe added to the kidney perfusion solution just prior to startingperfusion, or alternatively, may be added earlier and the resultingsolution stored prior to perfusion.

[0037] The present invention also relates to a process for preserving akidney for transplantation by perfusing the kidney with an amount of anitric oxide donor chemical in an amount sufficient to mimic enzymaticproduction of nitric oxide by NOS3 or NOS1, while preventing thegeneration of an excessive amount of nitric oxide by NOS2. This processmay occur with or without an additional reagent that could reduceglutathione back to its bioactive form in order to increase bioactiveglutathione. The organ to be transplanted may be perfused initially witha conventional static perfusion and then placed on a pulsatile perfusioncircuit with a solution containing a nitric oxide donor chemical and thechemical NOS2 inhibitor, with or without an additional reagent thatcould reduce glutathione back to its bioactive form to increasebioactive glutathione. Alternatively, the initial static perfusion maybe performed with a solution containing a nitric oxide donor chemicaland the chemical NOS2 inhibitor, with or without an additional reagentthat could reduce glutathione back to its bioactive form to increasebioactive glutathione. Thus, the present invention relates to a processfor preserving a kidney that includes the following steps:

[0038] 1. Flushing a kidney with a conventional flushing solution orwith a flushing solution that contains a nitric oxide chemical donor anda chemical inhibitor of NOS2 (with or without an additional reagent thatcould reduce glutathione back to its bioactive form) at a temperature ofbelow 10 degrees C.;

[0039] 2. Placing a cannula in the perfused kidney and mounting it in apulsatile perfusion cassette, connected to a pulsatile perfusionapparatus;

[0040] 3. Perfusing the kidney with a pulsatile perfusion solution thatcontains a nitric oxide chemical donor and a chemical inhibitor of NOS2(with or without an additional reagent that could reduce glutathioneback to its bioactive form) for up to 8 hours at a temperature of below10 degrees C.;

[0041] 4. Monitoring at least one physiological parameter which isindicative of organ function, including but not limited to: perfusionpressure, flow, vascular resistance, base excess, oxygen saturation,calcium, potassium, hematocrit, pO₂, pH, and bicarbonate.

[0042] The kidney is preferably maintained on a pulsatile preservationsystem at approximately 5 degrees C. until the time of transplant.However, the kidney may also be perfused at room temperature.

[0043] Another aspect of this invention involves the use of chemical NOdonors to maintain the function of organs while still in the deceaseddonor's body. In order to successfully recover organs fromnon-heartbeating donors (NHBD), from whom organs are recovered after theheart has stopped beating and all resuscitation methods have failed, thetrauma caused by ischemia or ischemia followed by reperfusion must beaddressed. Herein, it is proposed to address this problem bysupplementing with NO the flush solution used to initially perfuse theseorgans. In order to accomplish this successfully, the followingprocedure may be followed:

[0044] a) After death has been declared, the donor is asepticallyprepared for surgery, scrubbed and painted.

[0045] b) A femoral cut down is initiated, finding and isolating boththe femoral artery and vein in either the left or right groin dependingon the physician's preference.

[0046] c) The femoral artery is then cannulated with a 16 fr catheter,Porges MOP, and the femoral vein is cannulated with a 26 through 34frvenous return cardiothoracic vascular catheter.

[0047] d) The Porges catheter is primed with Lactated Ringer's solution(containing NaCl, sodium lactate, CaCl₂, and KCl in distilled water) orPlasmanate® solution (Plasma Protein Fraction [Human] 5%, USP; contains5 g selected plasma proteins [88% normal human albumin, 12% alpha andbeta globulins and not more than 1% gamma globulin] buffered with sodiumcarbonate and stabilized with 0.004 M sodium caprylate and 0.004 Macetyltryptophan) at room temperature (25 degrees C.), removing air, andthe solution is circulated through the vascular system of the donor toflush out the donor's non-oxygenated blood.

[0048] e) Once the donor's blood has been drained out, the drainagecircuit is then closed off and then a solution of Lactated Ringer's willhave nitric oxide, in the form of one or more of the chemical nitricoxide donors mentioned above, added to the flush solution along withoxygen.

[0049] The donor will be perfused with this solution for a minimum of 30minutes, at which point the solution will be cooled down to atemperature below 10 degrees C. The solution will continue to beperfused through the circuit until the donor is ready for the surgeon toremove the organs.

EXAMPLES

[0050] The following Examples illustrate in even greater detail specificembodiments of the invention. These Examples are intended to illustratethe practice and advantages of the invention, and are not intended tolimit the allowable scope of the invention in any manner whatsoever.

Example 1

[0051] Preparation of a Kidney Perfusion Solution Containing A NitricOxide Donor Chemical And A Chemical NOS2 Inhibitor

[0052] GSNO and L-NIL dihydrate hydrochloride were purchased from AlexisCorporation (San Diego, Calif.). Solution A was prepared by weighing out30 milligrams of GSNO powder and adding it to 1 milliliter of BelzerMachine Perfusion solution (Transmed, Elk Grove, Minn.), and theninjecting it into 1 liter of Belzer Perfusion solution while theperfusion solution was circulating through a Waters Instruments(Rochester, Minn.) kidney perfusion circuit (final concentration: 30milligrams/liters; 90 micromolar). Solution B was prepared by weighingout 187 milligrams of L-NIL dihydrate hydrochloride powder and adding itto 3.5 milliliters of Belzer Machine Perfusion solution, and theninjecting it into Solution A while the solution was circulating througha Waters Instruments kidney perfusion circuit (final concentration: 187milligrams/liter; 694 micromolar).

Example 2 Preservation of Porcine Kidneys Using Pulsatile PerfusionSolutions

[0053] Porcine kidneys were obtained following a warm ischemic time of30-45 minutes. The kidneys were flushed with 1 L of Lactated Ringer'ssolution at 5 degrees C., and then flushed with 1 L Viaspan™ solution(DuPont, Wilmington, Del.) at 5 degrees C. The kidneys were then storedat 5 degrees C. for 3-30 h, at which time the kidneys were placed on aWaters Instruments (Rochester, Minn.) kidney perfusion circuit andperfused with 1 L of Belzer Machine Perfusion solution (Transmed, ElkGrove, Minn.). During the perfusion period, the kidneys were kept at aconstant pressure of 40 mmHg. Eight kidneys were perfused with BelzerMachine Perfusion solution only, another eight kidneys were perfusedwith Belzer Machine Perfusion solution containing solution A (preparedas in Example 1), and a further eight kidneys were perfused with BelzerMachine Perfusion solution containing Solution B (prepared as in Example1).

[0054] Hourly samples (3 ml total) were taken from each kidney for atleast 5 hours, and in some cases 6 hours. Blood chemistry was analyzedusing a GEMStat analyzer (Instruments Laboratory, Boston, Mass.).Statistical analyses were performed using Sigmastat software (SPSS,Chicago, Ill.).

[0055] FIGS. 1-6 show how vascular resistance, flow, base excess, oxygensaturation, calcium and hematocrit were affected by the additionalpresence of a nitric oxide donor chemical (GSNO) or the combination ofGSNO and N-omega-imino ethyl lysine (L-NIL), a chemical inhibitorselective for the inducible isoform of the enzyme nitric oxide synthase.The greatest degree of improvement occurred with the combination of GSNO+L-NIL (Solution B). Several other parameters (pO₂, pH, potassium,bicarbonate and total CO₂) were not affected by the addition of GSNO andL-NIL. FIGS. 7-9 illustrate three of these parameters. TABLE 2 Summaryof the statistically significant effects of GSNO, with or without L-NILon explanted kidneys, relative to control Vascular Base Oxygen TreatmentResistance Flow Excess Saturation Ca²⁺ Hematocrit GSNO Lower Higher MoreEqual Higher Lower negative GSNO + Lower Higher More Lower Higher LowerL-NIL Negative (trend) (trend)

[0056] These changes are considered beneficial for explanted organfunction. For example, lower vascular resistance and higher flowindicate that a better supply of oxygen and nutrients can be provided tothe explanted organ. Lower oxygen saturation indicates that the organhas been more metabolic during the pulsatile perfusion procedure, andtherefore that this organ is more viable.

[0057] The treatment with GSNO and L-NIL appears to result in improvedkidney function compared to GSNO alone since oxygen saturation, which isindicative of metabolic activity of the kidney, was lower in this grouprelative to the control kidneys. In contrast, kidneys perfused withsolution A (GSNO only) did not differ statistically from the controlkidneys.

[0058] Decreased vascular resistance is an indication of improved organfunction. In this regard, L-NIL would be expected to increase vascularresistance by inhibiting the production of high levels of nitric oxidefrom NOS2 [see, for example, Petros et al., “Effects of a nitric oxidesynthase inhibitor in humans with septic shock,” 28 Cardiovasc.Res.34-39 (1994)]. Thus, the observed decrease in vascular resistance is anunexpected and surprising result.

We claim:
 1. A kidney perfusion solution comprising a) at least onegluconate salt; b) glutathione; c) a nitric oxide donor chemical; and d)a chemical inhibitor selective for the inducible isoform of the enzymenitric oxide synthase NOS2.
 2. The kidney perfusion solution of claim 1,wherein said nitric oxide donor chemical is at least one compoundselected from the group consisting of S-nitrosoglutathione;S-nitrosoalbumin; S-nitroso-N-acetyl-D,L-penicillamine;1,1-Diethyl-2-hydroxy-2-nitroso-hydrazine sodium;2,2′-(Hydroxynitrosohydrazono)bis-ethanimine;N-(2-Aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine;and their pharmaceutically acceptable salts, esters and derivatives. 3.The kidney perfusion solution of claim 2, wherein said nitric oxidedonor chemical is either S-nitrosoglutathione or S-nitrosoalbumin. 4.The kidney perfusion solution of claim 1, wherein said nitric oxidedonor chemical is present in an amount of 10 to 500 micromolar.
 5. Thekidney perfusion solution of claim 4, wherein said nitric oxide donorchemical is present in an amount of 20 to 125 micromolar.
 6. The kidneyperfusion solution of claim 1, wherein said chemical inhibitor is atleast one member of the group consisting of N-omega-imino ethyl lysine,N-omega-imino ethyl-L-ornithine, ([aminomethyl]benzyl)acetamidine,S-(2-aminoethyl)-isothiourea, aminoguanidine hydrochloride,S-ethyl-isothiourea, S-methyl-isothiourea and their pharmaceuticallyacceptable salts, esters and derivatives.
 7. The kidney perfusionsolution of claim 1, wherein said chemical inhibitor is present in anamount of 10 to 5000 micromolar.
 8. The kidney perfusion solution ofclaim 7, wherein said chemical inhibitor is present in an amount of from500 to 2000 micromolar.
 9. The kidney perfusion solution of claim 1,further comprising at least one superoxide scavenger or superoxideinhibitor.
 10. The kidney perfusion solution of claim 1, furthercomprising of a reagent capable of reducing oxidized glutathione. 11.The kidney perfusion solution of claim 1, wherein said reagent comprisesglutathione reductase.
 12. A process for preserving a kidney fortransplantation, comprising perfusing the kidney with an amount of anitric oxide donor chemical in an amount sufficient to mimic enzymaticproduction of NO by NOS3 or NOS1.
 13. A process for preserving a kidney,comprising: (a) flushing a kidney with a conventional flushing solutionor with a flushing solution that contains a nitric oxide chemical donorand a chemical inhibitor of NOS2, (b) placing a cannula in the perfusedkidney and mounting it in a pulsatile perfusion cassette, connected to apulsatile perfusion apparatus, (c) perfusing the kidney with a pulsatileperfusion solution that contains a nitric oxide chemical donor and achemical inhibitor of NOS2, (d) monitoring at least one physiologicalparameter which is indicative of kidney function.
 14. The process ofclaim 13, wherein said flushing solution further comprises an additionalreagent capable of reducing oxidized glutathione back to its bioactiveform.
 15. The process of claim 13, wherein said flushing step isperformed at a temperature of below 10 degrees C.
 16. The process ofclaim 13, wherein said perfusion solution further comprises anadditional reagent capable of reducing oxidized glutathione back to itsbioactive form.
 17. The process of claim 13, wherein said perfusing stepis performed for up to 8 hours at a temperature of below 10 degrees C.18. The process of claim 13, wherein said physiological parameter is amember selected from the group consisting of perfusion pressure, flow,vascular resistance, base excess, oxygen saturation, calcium, potassium,hematocrit, pO2, pH, and bicarbonate.
 19. A process for maintaining thefunctions of organs in a cadaver, comprising, A. isolating both thefemoral artery and vein in either the left or right groin of a cadaver;B. creating a flush circuit by cannulating the femoral artery with acatheter and cannulating the femoral vein with a venous returncardiothoracic vascular catheter, thereby creating a dreinage circuitwhich includes the vascular system of the cadaver, C. flushing asolution through said drainage circuit until the cadaver'snon-oxygenated blood has been removed from the cadaver; D. circulating aperfusion solution through said drainage system which includes a nitricoxide donor chemical and a chemical inhibitor selective for theinducible form of the enzyme nitric oxide synthase NOS2 for at least 30minutes, and E. circulating said perfusion solution at a temperaturebelow 10 degrees C. through said drainage system until at least oneorgan is removed from said cadaver.